Preparation and amplification of nucleic acids by means of magnetic particles

ABSTRACT

The invention relates to the preparation of a biological sample for performing verifications and examinations, wherein the aim of the invention is the creation of a method for preparing a biological sample having an improved PCR sensitivity compared to the reference standard having standard PCR without having to raise the cost thereof.

The invention relates to a preparation of a biological sample forperforming detections and investigations.

For the preparation of a biological sample, it is known from the priorart to first make the contents of a biological sample accessible (knownunder the term “lysis” or “digestion”), to selectively bind constituentsof the released contents of the biological sample on or to a solidsupport or carrier material (known under the term “binding”), toeliminate undesired constituents of the solid support or carriermaterial (known under the term “washing”) and to dissolve the desiredconstituents, namely nucleic acids, subsequently from the solid supportor carrier material (known under the term “elution”). At least onesought constituent, that is a defined part of a DNA or RNA strand, isfinally duplicated, that is amplified, for example by means of apolymerase chain reaction (PCR).

The PCR process consists of a number of, for example, 25-50 cycles,which are performed in a thermocycler. The following details areguideline values. Usually, a PCR must be optimized for the specificreaction.

Each cycle consists of three steps:

-   -   1. Denaturation (melting): First the double-stranded DNA is        heated to 94-96° C. to separate the strands. The hydrogen bonds,        which hold together the two DNA strands, are broken. In the        first cycle, the DNA is often heated for a relatively long time        (initialization) to ensure that both the starting DNA and the        primer have completely separated from one another and only        single strands are present. Some (so-called hot start)        polymerases must be activated by an even longer initial heating        phase (up to 15 minutes).    -   2. Primer hybridization (primer annealing): The temperature is        kept for about 30 seconds at a temperature that allows a        specific addition of the primers to the DNA. The exact        temperature is determined here by the length and the sequence of        the primers (or the appropriate nucleotides in the primer if        mutations are to be introduced by this=site-directed        mutagenesis). If the temperature chosen is too low, the primers        can under certain circumstances also add to non-100%        complementary sequences and thus lead to non-specific products        (“ghost bands”). If the temperature chosen is too high, the        thermal motion of the primers under certain circumstances is so        great that they cannot attach correctly so that no product        formation at all or only inefficient product formation occurs.        The temperature which largely excludes the two abovementioned        effects is normally 2-3° C. below the melting point of the        primer sequences; this usually corresponds to a temperature of        55-65° C.    -   3. Elongation (polymerization, extension, amplification):        Finally, the DNA polymerase fills up the missing strands with        free nucleotides. It begins at the 3′-end of the added primer        and then follows the DNA strand. The primer is not detached        again; it forms the start of the new single strand. The        temperature depends on the working optimum of the DNA polymerase        used (68-72° C.). This step lasts approximately 30 seconds per        500 base pairs, but varies as a function of the DNA polymerase        used. Customary thermocyclers cool the reaction batches down to        4-8° C. after completion of all cycles, so that a PCR can be set        up in the evening and the samples can be processed further on        the morning after.

In the first cycle, initially DNA single strands result, which arelonger in the 5′-direction than the target sequence. This can beexplained by the fact that only a starting point (primer), but not anendpoint, is fixed exactly. The termination of the strand synthesistakes place here at the latest by the strand separation in the followingdenaturation step. The DNA employed and the DNA strands just formed areavailable in the second cycle. In the former, the same process takesplace as in the first cycle. Primers now attach in the 5′-region to thenewly formed DNA single strands, which 3′ already end where they should.The strands now formed have no 5′-overlap, since the polymerase reads onthe template in the direction of the 3′-end. At the end of the secondcycle, there are thus products of the desired length for the first time.In the following cycles, the desired products replicate exponentially(as they themselves serve as a matrix for further strand syntheses),while the undesired long products (see products of the first cycle) onlyincrease linearly (only DNA employed serves as a matrix). This is thetheoretical ideal case; in practice, to a small extent, shorterfragments than the desired target DNA are also formed. These shortfragments especially accumulate in the late cycles, whereby usually onlyapproximately 30 cycles are run through to obtain all in all mainly DNAof the desired length and sequence.

A sample prepared in this way is identified subsequently to this, forexample, by means of agarose gel electrophoresis. A gel is prepared, forexample, by boiling agarose in a buffer, for example TBE buffer. Longthreads of agarose polymers are in this way crosslinked to give a gel.In general, auxiliaries are added to the gels even during preparationfor visualization of the separated molecules. In the case of DNA, theseare usually ethidium bromide. By means of agarose gel electrophoresis,nucleic acid strands (RNA or DNA) are separated according to their size,and their sizes are determined by comparison with strands of known size.The more highly the agarose is concentrated, the smaller are the poresthat are found in the gel. Gel electrophoresis functions like a sievefor molecules. An electrical field is used to pull the negativelycharged nucleic acid molecules through the gel matrix, it being possiblefor the smaller molecules to move more rapidly through the gel and thusseparation of the strands according to their size is made possible. Onaccount of added auxiliaries, the nucleic acids separated in this waycan generally be made visible with UV light. It can thus be determinedwhether a sought part of a DNA strand has been duplicated by the PCR andconsequently whether the sample originally contained the sought DNAstrands. Such detections are performed, inter alia, in paternity testsand in forensics.

For example, in the field of forensics generally only a restricted,small amount of sample material is available. In such cases it isimportant to obtain a high PCR sensitivity. The PCR sensitivity is ameasure of the duplication of the sought part of a DNA strand. The moresensitive the PCR, the better and more rapidly the corresponding DNAstrand is duplicated.

An automated process for the preparation of a biological sample, whichcomprises the steps lysis, binding, washing, elution and PCR, can beinferred from the European patent application having the official filereference EP08151152.9: A biological sample is transferred to acontainer that contains a filter consisting, for example, of a silicagel. Below the filter is provided an inlet or outlet for liquid. First,a biological sample is digested using a lysis buffer, in particular inthis container. After the digestion of the sample, the lysis buffer issucked off through the filter. It is simultaneously achieved therebythat the nucleic acid released by digestion binds to the filter. Thebinding of the nucleic acid to the filter is increased by a subsequentaddition of ethanol to the vessel. With the aid of wash buffers, it isthen washed to remove fats, lipids and proteins. After washing, thebound nucleic acid is dissolved from the filter with the aid of elutionliquid. The elution liquid is supplied together with the nucleic acidsnow situated therein to a PCR chamber in order to carry out the desiredpolymerase chain reaction therein.

From the European patent application with the official file referenceEP08151152.9, it furthermore emerges to separate the result of the PCRelectrophoretically with the aid of an agarose gel and to stain theseparated nucleic acid fragments in an ethidium bromide-containing waterbath. By excitation with UV light, the stained nucleic acid fragmentsbecome visible and are thus detected. Binding takes place based onchaotropic chemistry. It is then bound under high salt (that is asolution having a high salt concentration). An ethanol elution takesplace with low salt, that is a solution having a low salt concentration.If, on the other hand, an anion exchanger is employed, the binding takesplace under low salt and elution under high salt.

The described preparation of a biological sample, which comprises saidsteps lysis, binding, washing, elution and PCR, is called a referencestandard or else gold standard. A high PCR sensitivity can be achievedwith the gold standard. A small amount of sample material then sufficesto be able to adequately duplicate the DNA strand sought. In general,the reference standard comprises a standard nucleic acid preparation(with the steps lysis, binding, washing and elution) and subsequently astandard PCR and not the markedly more expensive, but also markedly moresensitive nested PCR, since the standard PCR for carrying out thedesired detections generally suffices. PCRs are in principle susceptibleto contamination by inhibitors, which can decrease the efficiency of thereaction. The advantage with a nested PCR is that even if the first PCRexhibits slight inhibition—a high sensitivity can be achieved by thesecond PCR which then generally proceeds without inhibition problems,despite primary inhibition in the first PCR. If, on the other hand, an“individual” PCR (reference standard), for example a qPCR, exhibitscontamination by inhibitors, then the efficiency and thus also thesensitivity of the reaction is decreased. A problem, however, in thecase of a nested PCR in comparison to the standard PCR, is theoccurrence of cross-contamination. As a result of aerosols and other“accidents”, the danger of false-positive results is very high in thecase of the nested PCR.

A preparation of a biological sample according to the gold standard canfurther be inferred from the printed specification WO 93/11221. It isknown from this to digest biological samples by use of enzymes, such as,for example, proteinase K, lysozyme and detergents such as SDS, Brij,Triton X-100, Tween 20, DOC and chemicals such as sodium hydroxide,guanidine hydrochloride and guanidine isothiocyanate. After thedigestion and removal of undesired cell debris, the nucleic acid to beisolated is bound to an anion exchanger. It can further be inferred fromthe printed specification that after the binding, substances to beseparated off are first removed by washing, in order then to detachagain the bound nucleic acids with an elution buffer of high ionicstrength. It can further be inferred from this prior art that thefurther process operations are only possible using buffer conditionswhich exhibit lower ionic strengths. In particular, before theperformance of a PCR, the nucleic acid must first be desalted followingthe elution, which disadvantageously necessitates a correspondinglyincreased outlay. According to the printed specification WO 93/11221, acommercially available material can be selected as an anion exchanger,which allows binding of the nucleic acid to be isolated under therespective preparation conditions. The anion exchangers known from WO93/11221 are preferably surface-modified carriers consisting of amatrix, preferably consisting of agarose, dextran, cellulose,acrylamide, polyvinyl alcohol, polystyrene, glass, alumina, titaniumdioxide, zirconium dioxide or silica gel, such as, for example,DEAE-Q-Sepharose®, Q-Sepharose®, DEAE-Sephadex®, DEAE-Toyopearl®,Amberlite®, Nukleogen®. The anion exchangers can be porous carriermaterials having an internal surface of high capacity suitable forinteraction or non-porous carrier materials, which only enters into aninteraction with the mixture to be separated on the external surface.Very particularly preferably, the anion exchanger is a material based onsilica gel, which has a particle size of 1 to 250 μm, preferably 10 to50 μm and very particularly preferably 15 to 25 μm and a pore diameterof 1 to 2500 nm, preferably 10 to 500 nm, particularly preferably 100 to400 nm. A material having high surface charge and high binding capacityfor nucleic acids has in particular proven to be an anion exchangematerial.

The modification of the silica gel is performed according to WO93/11221, preferably by silanization of the carrier material, such asdisclosed, for example, in EPA 83 901 065, DE-A-39 35 098 and U.S. Pat.No. 5,057,426, In EP-A 83 901 065, for example,gamma-glycidyloxypropyltrimethoxysilane and N,N-dimethylaminoethanol areused for the modification of the carrier material.

If the PCR sensitivity obtained according to the gold standardcomprising the standard PCR does not suffice, the markedly moresensitive nested PCR is performed, in which two PCR reactions areconnected one after the other. An aliquot of the PCR product from thefirst amplification serves as a matrix for the second PCR. In this, ashorter DNA fragment is amplified by a second primer pair, which bindsto sequence regions within this matrix (nested primer). The advantage ofthe nested PCR compared to the standard PCR is a sensitivity increasedby 2-3 powers of ten paired with increased specificity, as for thenested PCR product only the product of the first amplification can serveas a matrix. The smallest traces of DNA can also be detected using thismethod and diagnostic aims can be made accessible. With optimaladjustment of a nested PCR, even 1 to a few matrixes suffice foramplification depending on the target sequence and object. In comparisonto the standard PCR, however, additional process steps mustdisadvantageously be performed. If the PCR sensitivity of a samplepreparation according to the gold standard does not suffice with thestandard PCR, a process would then be desirable which makes possible animproved PCR sensitivity, without having to increase the necessaryoutlay.

Indeed, there are alternative processes which lead to good PCRsensitivities without an outlay having to be made for this, whichcorresponds to the gold standard with a nested PCR. Thus, for example,the printed specification WO 92/17609 A1 discloses a process for thedetermination of target cells, in which the cells are first bound intactto magnetic grains—known under the term “magnetic beads”—with the aid ofantibodies and thus enriched. Such processes, however, are very specificand can therefore only be employed to a relatively restricted extent.

The object of the invention is the creation of a simple process for thepreparation of a biological sample having high PCR sensitivity.

The object is achieved by a process having the features of claim 1.Advantageous embodiments result from the subclaims.

To achieve the object, a process for the preparation of a biologicalsample comprises the steps lysis, binding, optional washing and atwo-stage amplification. During the first amplification, the sought DNAstrands are amplified relatively unspecifically. The relativelyunspecifically amplified DNA strands thus obtained are subsequentlyamplified specifically. The two-stage amplification is in particular anested PCR. Differing from the gold standard, the elution and the inmany cases subsequent desalting of the solution resulting from theelution are unnecessary. Indeed, a two-stage amplification such as thenested PCR in comparison to the standard PCR necessitates an additionaltechnical and temporal outlay. This is compensated, however, in theprocess according to the invention by the elution in addition todesalting being unnecessary. Altogether, the necessary outlay accordingto the invention corresponds to the outlay that is necessary in themaintenance of the gold standard using standard PCR. However, incomparison to the gold standard comprising a standard PCR, the PCRsensitivity can be markedly increased. Altogether, the ratio of outlayto benefit can therefore be increased using the process according to theinvention. The process according to the invention can comprise the stepsand devices known from the prior art provided these do not counteractthe performance of the process according to the invention. Thus, forexample, the lysis buffer or anion exchanger mentioned at the outset canbe used Thus the lysis in addition to binding can take place onpreferably magnetic silica particles in a known manner by means ofchaotropic salts and by means of ethanol. The lysis can be assistedmechanically, in fact preferably by ultrasound, or by stirring with theaid of a magnetically driven stirring rod, in order to mechanicallyassist a digestion rapidly and simply.

Instead of a PCR, other amplification processes can also be performed,thus, for example, WGA (whole genome amplification) or reversetranscription. However, a nested PCR is preferably performed, or anested PCR in which the first PCR comprises a one-step RT PCR.

PCR, however, is the most robust process for the two-stage amplificationprocess. For the person skilled in the art, it is possible in principleto carry out the first and the second nucleic acid amplification alsousing processes that are not based on the use of a heat-stablepolymerase and multistage temperature steps (that is on PCR). Suchprocesses for the amplification of DNA area “Helix-dependentAmplification” (HDA); “Recombinase Polymerase Amplification” (RPA);“Sequence-Specific Rolling Circle Amplification (RCA); “Loop-mediatedIsothermal Amplification” (LAMP). For the amplification of RNA, methodssuch as “Signal Mediated Amplification of RNA Technology” (SMART) or“Nucleic acid sequence-based amplification” (NASBA) can also be usedalternatively to simple reverse transcription. Of course, it is alsopossible for the person skilled in the art to combine these and otherknown processes for amplification according to need. The first and thesecond amplification can also be designed as multiplex amplifications.

Specific amplifying means that in the ideal case only one amplicon isduplicated. “Unspecific amplifying” means a simultaneous and unspecifiedamplification of a number of gene sequences by an incompletehybridization of the primers, i.e. the primers also bind incompletelywith “mismatch” in the first, cycles of the PCR to the starting nucleicacid and are lengthened by the polymerase. These “wrong” amplificatescan function again as templates in the course of the PCR (the primersthen bind perfectly) and are then amplified—this then manifests itselfin the form of byproducts in the PCR.

Preferably, the binding takes place by means of anion exchange, sinceeven during the lysis binding can take place. This embodiment of theinvention contributes to the fact that the process can be performedeasily in a closed, very small microfluidic system. According to theprior art, the binding by means of anion exchange is indeed linked tothe disadvantage that the elution necessitates the use of salts of highconcentration, which makes desalting necessary following the elution.Since elution is unnecessary according to the invention, a subsequentdesalting is also unnecessary.

In order to be able to supply the bound nucleic acid suitably to anested PCR, the bound nucleic acid is separated after the binding in oneembodiment of the invention, by, for example, centrifuging particles towhich the nucleic acid is bound. The supernatant, that is the bufferlast used or the solution last used, is discarded. If, for example, itwas previously washed, the wash buffers are thus removed by finaldiscarding of the supernatant. The PCR sensitivity is increased by theseparation. For example, a sample contains 10³ bacteria or viruses.According to the prior art, a lysate of typically 1.5 ml total amount isobtained from this. Of this, however, a PCR can be performed only with 1to 3 microliters. The consequence of this is that only 0 to 1 copy(ies)can get to a PCR 1. This small amount can lead to sensitivity problems.By means of the concentration according to the invention, it is achievedthat approximately quantitatively 10³ copies of the PCR 1 are suppliedand thus contribute to achieving the surprisingly high sensitivity.

In one embodiment of the invention, the aforementioned bodies aremagnetic particles (“magnetic beads”). The desired separation orconcentration can thereby be performed particularly rapidly, namely, forexample, with devices known from the prior art provided for this. Tothis end, for example, a tube used for carrying out the preceding stepsis inserted in a commercially obtainable device. The permanent magnetspositioned thereon attract the magnetic particles to the wall of thetube. The supernatant can thus be safely removed after such a magneticseparation. The supernatant is in turn discarded. Using the separatedconstituents, which comprise the magnetic particles in addition to thenucleic acids bound thereto, a PCR 1, for example, that is the firststage of a nested PCR, is performed. This embodiment of the inventionfurther contributes to the fact that the process can be performed in asimple manner in a closed, microfluidic system. A magnet is then locatedin a first PCR chamber, subsequently also called a central magnet. Thiscentral magnet is larger than the diameter of exit channels or else theexit channels are separated from the first PCR chamber by a sieve or afrit. If the magnetic particles with the nucleic acids bound thereto arenow transported into the first PCR chamber with the aid of a liquid, themagnetic particles, that is the magnetic beads, stick to the magnet. Ifthe liquid is aspirated from the first PCR chamber, the desiredseparation thus takes place. The channel diameters of the channelsleading off or the pore width of the frit or of the sieve are chosensuch that the central magnet located in the chamber, to which no nucleicacids are bound, is restrained. Since only a relatively large magnetwith magnetic grains or beads adhering thereto must be restrained, thedanger also does not exist that exit channels or sieves can be addeddisadvantageously. In principle, the process can also be performed,however, using non-magnetic grains or beads, which are then separated bya sieve in the PCR chamber from the solution or liquid with which thebeads or grains were previously transported into the first PCR chamber.

Following a performed PCR 1 of a nested PCR, separation again takesplace, which in the case of magnetic particles employed takes placeparticularly simply with the aid of a magnet. The supernatant containsamplicons, that is DNA strands, which have been duplicated by the PCR 1.An aliquot of the supernatant, that is a part of the supernatant, isadded to the PCR 2. The nested PCR is continued here in a manner knownfrom the prior art.

In one embodiment of the invention, the process is performed completelyor at least mainly in a closed system, which already comprises the PCRreagents needed, namely in particular in dried form. It is thenessential that the nested PCR or an equivalent two-stage amplificationprocess is especially performed in a closed system, which can inparticular then be realized technically without difficulty in adisturbance-free manner if the nucleic acids are bound to magneticparticles. Indeed, a nested PCR is very sensitive, but the nested PCR,however, has a disadvantage, especially because of its highsensitivity—it is very susceptible to contamination. As a result ofunwanted spread of amplicons and their de novo amplification,false-positive results can easily occur. In order to guard against thesedifficulties, it is advantageous to carry out the PCR reaction in aclosed system with PCR reagents contained therein. False-positiveresults can thus be minimized.

The process can even be performed in a microfluidic system, which thenminimizes even the space requirement.

In one embodiment of the invention, the lysis buffer employed has a saltcontent of in total less than 50 millimolar, that is 50millimoles/liter. It has turned out that by observing this limit the PCRsensitivity can be markedly increased.

The pH of the lysis buffer used is preferably adjusted to values betweenpH 7.5 to 9.5. This also increases the PCR sensitivity. In order tostabilize the pH in the manner mentioned, commercially obtainablebuffers tris, hepes and/or mops are preferably employed, in particularwith a concentration of 10 to 20 millimolar, in order to achieve goodPCR sensitivities. The aforementioned designations are abbreviationswhich are familiar to the person skilled in the art.

As detergents, in one embodiment of the invention non-ionic detergentsare employed for performing the lysis. Suitable nonionic detergents aremarketed commercially under the trade names Tween®, Nonidet P-40 orBrij®. This selection also makes it possible to achieve good PCRsensitivities.

The concentration of nonionic detergents is preferably 0.1 to 0.4% byvolume.

Optionally, polyvinylpyrrolidone (abbreviated as PVP) and namely inparticular from 0.05 up to 0.1% by volume is added to the lysis buffer.Inhibitors are bound by PVP and good PCR sensitivity is obtained in afurther improved manner.

A simple process for the preparation of a biological sample having analways still relatively high PCR sensitivity can also be achieved bybinding the nucleic acid obtained by digestion to particles, separatingthe particles with the nucleic acids bound thereto and amplifying thebound nucleic acids. The amplification can then also take place by meansof a conventional process such as, for example, a qPCR, that is aprocess in which a quantitative evaluation takes place only aftercompletion of the PCR. A relatively high sensitivity is especiallyachieved if the lysis buffer is suitably chosen. A suitably chosen lysisbuffer in particular comprises the features of a lysis buffer indicatedin subclaims. However, other isothermal amplifications can also beperformed.

The invention is illustrated in more detail below with the aid ofexamples.

Anion exchange particles (namely RSE anion exchange beads or grains) forthe binding of nucleic acid were prepared as follows, the preparationbasis of these particles being described in the patent application: WO2007/065933 A1:

As a preparation, 302 ml each of SPAN 85 are dissolved in 13 l of Norpar12 and 1.9 l of viscous paraffin. Subsequently, 112.5 ml of EGDMA and261 ml of GMA, which have been freed from inhibitors beforehand, areadded to a 2000 ml Nalgene bottle or a 2000 ml beaker, 370 ml ofethylene glycol, 11.25 g of AIBN and 279 g of Bayoxide E 5706 are addedand the mixture is homogenized for 2 minutes at the highest setting inan IKA Ultra Turrax. A quarter of the Norpar solution is added to a 5000ml plastic vessel in four portions and the vessel is connected to ahomogenizer. One quarter each of an iron oxide suspension is added atthe lowest setting and the mixture is homogenized for 60 seconds underfull power. The emulsion is then in each case transferred to a 20 lreactor with a reflux condenser, KPG stirrer and a line for passing gasthrough at 250 rpm and the speed of rotation of the stirrer is increasedto 300 rpm. The iron oxide emulsion in organic solvent is thensubsequently added slowly, the mixture is degassed for 5 minutes bypassing through nitrogen, the stirrer is speeded up to 350 rpm, and themixture is then stirred overnight at 350 rpm. Heating is already begunwhile passing through gas, then the mixture is kept at 70° C. for onehour, then overnight at 80° C.

The next morning, the quantities of organic solvent are filtered offthrough a suction filter with a polyethylene frit and the oil residuesare stored. The magnetic residue is then washed three times withacetone, additionally waiting about 10 minutes after acetone additionhere before filtering off with suction. The residue is then taken up incompletely demineralized water, washed three times with water, andwashed once again with acetone and twice with abs. ethanol, and thesolvents are separated off here using magnetic separation, stirring uphere and allowing the solvent to act for 5 minutes. In the first waterwashing step, ultrasound is allowed to act on the vessels for 10 minutesin an ultrasonic bath and the separation is then continued. After thelast water step and the last three acetone and ethanol steps, the liquidis basically separated off, or the particles are allowed to stand in thelast ethanol solution. In order to determine the yield, a portion isremoved from the last suspension and dried for determination of thesolids content. The finished product has an average particle sizedistribution of 10 μm.

First, a polymer suspension containing 2.5 g in ethanol is added to aglass filter funnel, porosity 4 and washed four times with anhydrousdiglyme or toluene. The residue is taken up in 50 ml of an almost 10%strength solution of bis-tris or diisopropylaminoethylamine in anhydrousdiglyme or toluene and transferred to a 100 ml three-necked flask. Areflux condenser is then attached to the flask, the latter is brieflydegassed twice and the mixture is then allowed allowed to reactovernight (16 h) at 100 rpm and 120° C. The next morning, the polymersare washed four times with completely demineralized water, three timeswith ethanol and the polymers are then stored under ethanol.

A further type of functionalized anion exchanger magnetic particle (AX1)is synthesized as follows.

A suspension of MagAttract “G” (from BioSprint DNA blood kit 96, QIAGEN,item #940057) is employed and washed four times with Millipore water.The supernatants are separated off here by means of magnetic separationand discarded. Subsequently, 2.5 g of the dried magnetic particles areresuspended in 25 ml of dry γ-glycidoxypropyltriethoxysilane (Aldrich,item #4401.67). The mixture is then briefly degassed on a rotaryevaporator and aerated with nitrogen. Subsequently, the bath is heatedto 140° C. and the sample is allowed to react on the rotary evaporatorfor eight hours. The product is then separated magnetically and thesupernatants are washed four times with dry dioxane. The magnetic silicagel is then suspended in 25 ml of dry dimethylaminoethanol and treatedwith 250 μl of boron trifluoride etherate. Subsequently, the productmixture is heated under reflux for 24 hours on the rotary evaporator andmagnetically separated after cooling, the supernatants being discarded.It is then washed repeatedly with dioxane, methanol and diethyl etherand dried at 50° C. in a vacuum drying oven.

In order to determine the sensitivity of the process according to theinvention, a real-time PCR is performed as PCR 2 in the followingexamples, with which the DNA obtained is quantified.

The real-time PCR is a duplication method for nucleic acids, which isbased on the principle of the conventional polymerase chain reaction(PCR), and additionally makes possible the quantification of the DNAobtained. The quantification is performed with the aid of fluorescencemeasurements, which are determined during a PCR cycle. The fluorescenceincreases proportionally with the amount of the PCR products. At the endof a run (which consists of several cycles) the quantification in theexponential phase of the PCR is performed by means of fluorescencesignals obtained. Only in the exponential phase of the PCR (which lastsfor only a few cycles in a run) is the correct quantification possible,since during this phase the optimal reaction conditions prevail. Thismethod thus differs from other quantitative PCR methods (qPCR), whichperform a quantitative evaluation (e.g. competitive PCR), usuallyinvolving a gel-electrophoretic separation of the PCR fragments, onlyafter completion of the PCR.

In the first phase of the amplification of a PCR, the template amount isrestricted and the probability that template, primer and polymerase meetis suboptimal, while in the third phase of the amplification the amountof the products (DNA, pyrophosphate, monophosphate nucleotides)increases such that inhibition by this occurs, product fragmentshybridize with one another more frequently, the substrates are slowlyconsumed and finally the polymerases and nucleotides are slowlydestroyed by the heat. An exponential and therefore quantifiableincrease is found only in the phase in between. Exponentially, a PCRremains at 12 to 400 starting copies for about 30 cycles, at 200 to 3200for 25 cycles and at initially 3200 to 51,200 for at most 20 cycles. Inorder always to be able to measure at the start of the exponentialphase, frequently the CT value (threshold cycle) or the Cp value(crossing point) is used, which describes the cycle in which thefluorescence for the first time increases significantly above thebackground fluorescence.

In the course of a first exemplary embodiment, the bacterium Escherichiacoli was diluted in blood and investigated. Experiments were performedusing the different anion exchange speeds mentioned in order toinvestigate whether the choice of the anion exchange beads influencesthe result with differing surface modification.

The following materials were employed:

-   -   AX1 anion exchange beads    -   RSE anion exchange beads    -   blood from blood bag    -   lysis buffer GE (01% PVP MW 10000; 0.45% Tween-20; 0.45% NP 40;        10 mM tris/Cl pH 7.5; 1 mM EDTA)    -   Escherichia coli 0157:H7 (ATC 700728; apathogenic strain)        -   dilutions: 10⁸/ml; 10⁷/ml; 10⁶/ml; 10⁵/ml;

PCR primers EOF: ATGCTACCCCTGAAAAACTC EOR: CGCTTGAACTGATTTCCTCEFwd: CGATGATGCTACCCCTGAAAAACT ERev: TATTGTCGCTTGAACTGATTTCCTCEPro: “6-Fam”-CGTTGTTAAGTCAATGGAAAACCTG-BHQ1

The experiment was performed according to the following protocol:

In each case 100 μl of blood and 10 μl of a dilute E. coli overnightculture are filled into various 1.5 ml microtubes, that is into small,tubular vessels. The dilutions were 10⁸/ml; 10⁷/ml. 10⁶/ml; 10⁵/ml. 1 mlof buffer GE and 20 μl of proteinase K and 10 μl of lysozyme (10 mg/ml)were filled into the microtubes.

20 μl of anion exchange magnetic particles (50 mg/ml) were added to themicrotubes, namely, depending on batch, either AX1 or RSE magneticparticles. An incubation was subsequently performed by treating themicrotubes for 30 min on an Eppendorf thermomixer at 56° C. and 1400revolutions per minute. The incubation was then continued, namely for 10min on the Eppendorf thermomixer at 80° C. and 1400 revolutions perminute. The sample is digested and bound by this treatment.

The tubes are then inserted into a magnetic separation device. For 20sec, the magnetic particles are allowed to deposit on the wall of thetube by the action of the magnetic field of the permanent magnets of thedevice. The supernatant is now pipetted off and discarded. Subsequently,the magnetic particles are washed twice with buffer GE. For this, 800 μleach of buffer GE are pipetted into the tube and the magnetic particlesediment is resuspended by brief vortexing. Next, a magnetic separationis performed and the supernatant is again discarded.

The magnetic particle sediment is now resuspended by brief vortexing in100 μl of double-distilled, that is twice-distilled, water andtransferred to a PCR tube. The supernatant is removed carefully andquantitatively. The magnetic beads are resuspended with 50 μl of PCRsolution and the first PCR is started and performed as follows:

25 μl of Hot Star Tag Master Mix (QIAGEN), 1 μl of EOF (100 μM), 1 μl ofEOR (100 μM) and 23 μl of water are added. After activation of the Tagpolymerase for the first time by incubation at 95° C. for 15 min, atotal of 20 temperature cycles are performed. A cycle comprises thefollowing steps:

-   -   Denaturation 15 s; 95° C.    -   Annealing 30 s; 52° C.    -   Extension 15 s; 72° C.

After PCR1, the PCR tube is inserted onto a magnetic separation rack and5 μl of supernatant are transferred to a fresh PCR tube.

The second PCR, namely a quantitative real-time PCR, is begun with anaddition of 20 μl of PCR 2 reagents. The following reagents areemployed:

12.5 μl of QIAGEN QuantiTect sample PCR Mastermix, 0.1 μl of EFwd; 0.10μl of ERev; 0.05 μl of EPro and 7.25 μl of double-distilled water. Afterthe Tag polymerase activation for 5 min at 95° C., a total of 40 cyclesare performed. A cycle comprises the following steps: (denaturation 15s; 95° C.; extension 60 s; 60° C.).

As a comparison experiment, a preparation according to the gold standardwas performed with a standard PCR with according to the commerciallyobtainable “QIAamp DNA Blood Mini Kit”, 10 μl of E. coli dilution wereprepared together with 100 μl of blood according to the standardprotocol and eluted with 100 μl of elution buffer.

The result of the quantitative PCR for the detection of E. coli DNAaccording to example 1 using different numbers of bacteria in thepreparation is shown in FIG. 1 and compared with the result that wasobtained by the standard preparation using elution and standard PPCR.The ct values obtained according to the invention were around about twounits below the ct values that were obtained on account of the standardprocess. Since the sensitivity is all the better, the lower the ctvalue, despite comparable outlay the sensitivity was thus improved bythe process according to the invention. The test series furtherillustrate that using the process according to the invention, a goodlinearity can be achieved that reflects the dynamic range of the PCRdetection system. In this respect too, a very good result was thusachieved. Finally, the result illustrates that the result does notdepend substantially on the choice of the anion exchange beads.

In the course of a second exemplary embodiment, the bacteriumEscherichia coli was diluted in SurePath medium and prepared andinvestigated as follows according to the invention and—for the purposeof comparison—according to the prior art.

Material Used

-   -   AX1 anion exchange beads    -   RSE anion exchange beads    -   blood from blood bag    -   lysis buffer GE (01% PVP MW 10,000; 0.45% Tween-20; 0.45% NP 40;        10 mM tris/Cl pH 7.5; 1 mM EDTA)    -   Escherichia coli 0157:H7 (ATTC 700728; apathogenic strain)    -   dilutions: 10⁸/ml; 10⁷/ml; 10⁵/ml; 10⁵/ml;

PCR primers EOF: ATGCTACCCCTGAAAAACTC EOR: CGCTTGAACTGATTTCCTCEFwd: CGATGATGCTACCCCTGAAAAACT ERev: TATTGTCGCTTGAACTGATTTCCTCEPro: “6-Fam”-CGTTGTTAAGTCAATGGAAAACCTG-EHQ1

The following were prepared according to the following protocol

-   -   100 μl of blood and 10 μl of E. coli overnight culture are added        in various dilutions to a 1.5 ml microtube    -   addition of 1 ml of buffer GE and 20 μl of proteinase K and 10        μl of lysozyme (10 mg/ml)    -   addition of 20 μl of MagBeads    -   incubation: 30 min on Eppendorf thermomixer at 56° C. and 1400        rpm    -   incubation: 10 min on Eppendorf thermomixer at 80° C. and 1400        rpm    -   magnetic separation and discarding of the supernatant    -   wash Mag Beads 2X with buffer GE (800 μl each; briefly vortex;        magnetic separation and discarding of the supernatant)    -   resuspend MagEead sediment in 100 μl of double-distilled water        and transfer to a PCR tube    -   remove supernatant carefully and quantitatively    -   resuspend beads with 50 μl of PCR solution and start PCR1

Nested PCR1

-   -   25 μl of Hot Star Tag Master Mix (QIAGEN)    -   1 μl of EOF (100 μM)    -   1 μl of EOR (100 μM)    -   23 μl of water    -   Tag activation 15 min; 95° C.

Cycle:

-   -   denaturation 15 s; 95° C.    -   annealing 30 s; 52° C.    -   extension 15 s; 72° C.    -   number of cycles 20    -   After PCR1: place PCR tube on magnetic separation rack and        transfer 5 μl to a fresh PCR tube    -   carrying out PCR2 (quantitative real-time PCR) by addition of 20        μl of PCR 2 reagents:        12.5 μl of QIAGEN QuantiTect sample PCR Mastermix, 001 μl of        EFwd; 0.10 μl of ERev; 0.05 μl of EPro, 7.25 μl of        double-distilled water; cycling: Tag activation 5 min; 95° C.;        40 cycles [denaturation 15 s; 95° C.; extension 60 s; 60° C.]

Control experiment according to the prior art: QIAamp [10 μl of E. colidilution in 100 μl of blood were prepared according to standard protocoland eluted with 100 μl]. “QIAamp nested:” 5 μl of the eluate wereemployed in PCR1 and of this 5 μl were again employed in PCR2: “QIAampdirect 5 μl of the eluate were employed in PCR 2”.

FIG. 2 shows the result of the quantitative PCR for the detection of E.coli DNA from example 2 with different numbers of bacteria in thepreparation. From the result thus achieved, it is surprising that eventhe sensitivity was achieved that was achieved by a sample preparationaccording to the gold standard using a nested PCR (QIAamp nested). Inturn, a great linearity was also obtained. The result from example 1 wasfurther confirmed that it was possible to markedly increase thesensitivity compared to the standard process using the standard PCR(QIAamp direct) without having to increase the outlay for this.

In the course of a third exemplary embodiment, SIHA cells were preparedand investigated in SurePath medium.

The materials employed were

-   -   Cultured SIHA cells (human cervical carcinoma cell line; 2-3        copies of HPV16/cell; ATCC: HTB 35).

The cells were cultured in cell culture flasks by means of standard cellculture protocols. After washing with PSB, the cells were trypsinizedand the cell count was determined under the microscope. 10⁶ cells ineach case were aliquotted and sedimented by centrifugation. After theremoval of the supernatant, the cells were frozen as a sediment. Forpreparation, the cells sediments were resuspended by addition ofSurePath medium. Further dilutions were in each case prepared usingSurePath medium.

-   -   AX1 anion exchange beads    -   RSE anion exchange beads    -   Lysis buffer GE (01% PVP MW 10,000; 0.45% Tween-20; 0.45% NP-40;        10 mM tris/Cl pH 7.5; 1 mM EDTA)

PCR primers 16oFWD CACCAAAAGAGAACTGCAATG 16iFWD GGAGCGACCCAGAAAGTTACCAC16oREV GGATTCCCATCTCTATATACTA 16iREV GCATAAATCCCGAAAAGCAAAGTCA 16PROAGAATGTGTGTACTGCAAGCAACAG[BHQ-FAM]

The procedure was as follows according to the following protocol:

-   -   500 μl of SurePath medium containing SIHAs cells in various        dilutions are added to a 2.2 ml microtube    -   addition of 1 ml of buffer GE and 20 μl of proteinase K    -   addition of 20 μl of MagBeads    -   incubation: 30 min on an Eppendorf thermomixer at 56° C. and        1400 rpm    -   incubation: 10 min on an Eppendorf thermomixer at BOC and 1400        rpm    -   magnetic separation and discarding of the supernatant    -   wash Mag Beads 2× with buffer GE (800 μl each; briefly vortex;        magnetic separation and discarding of the supernatant)    -   resuspend MagBead sediment in 100 μl of double-distilled water        and transfer to a PCR tube    -   carefully and quantitatively remove supernatant    -   resuspend beads using 50 μl of PCR solution and start PCR1

Nested PCR1:

-   -   -   25 μl of Hot Star Tag Master Mix (QIAGEN)        -   1 μl of 16oFWD (100 μM)        -   1 μl of 16oREV (100 μM)        -   23 μl of water

Cycling

-   -   -   Tag activation 15 min; 95° C.        -   denaturation 15 s; 95° C.        -   annealing 30 s; 52° C.        -   extension 15 s; 72° C.        -   number of cycles 20

    -   After PCR1; place the PCR tube on the magnetic separation rack        and transfer 5 μl to a fresh PCR tube

    -   performing PCR2 (quantitative real-time PCR) by addition of 20        μl of PCR 2 reagents:

    -   12.5 μl of QIAGEN QuantiTect sample PCR Mastermix, 0.1 μl of        16iFWD; 0.10 μl of 16iRev; 0.05 μl of 16iPro, 7.25 μl of        double-distilled water; cycling: Tag activation 5 min; 95° C.:        40 cycles [denaturation 15 s; 95° C.; extension 60 s; 60° C.]

    -   Comparison experiments: QIAamp [500 μl of SurePath medium with        SIHAs cells prepared, according to standard QIAamp protocol and        eluted with 100 μl). “QIAamp nested” 5 μl of the eluate were        employed in PCR1 and of this again 5 μl in PCR2.

FIG. 3 shows the result of the quantitative PCR for the detection ofHPV16 from SIHA cells from exemplary embodiment 3 using different SIHAcell counts in the preparation.

Conclusion: The strategy of “nested PCR bead cycling” for thepreparation of HPV16 from SurePath medium shows a high linearity ofdetection of cells employed. Up to 100 cells can be safely detected. Theprocess according to the invention is thus also suitable for HPVdiagnosis.

In the course of a fourth exemplary embodiment, the binding andamplification were performed by means of silica MagBeads.

The following were employed as materials:

-   -   MagAttract suspension G; QIAGEN    -   MasB: MagAttract suspension B; QIAGEN    -   SurePath medium (BD)    -   lysis buffer G (3 M GITC; 20% NP-40)    -   Escherichia coli 0157:H7 (ATTC 700728; apathogenic strain)        -   dilutions: 10⁸/ml; 10 ⁷/ml; 10 ⁶/ml; 10 ⁵/ml

PCR primers EOF: ATGCTACCCCTGAAAAACTC EOR: CGCTTGAACTGATTTCCTCEFwd: CGATGATGCTACCCCTGAAAAACT ERev: TATTGTCGCTTGAACTGATTTCCTCEPro: “6-Fam”-CGTTGTTAAGTCAATGGAAAACCTG-BHQ1

The procedure was according to the following protocol:

-   -   500 μl of SurePath medium and 10 μl of E. coli overnight culture        are added in various dilutions to a 1.5 ml microtube    -   addition of 500 μl of buffer G and 20 μl of proteinase K        (QIAGEN)    -   incubation: 20 min 56° C.    -   addition of 500 μl of isopropanol and 20 μl of silica magnetic        particles (MasG or MasB)    -   shaking at 1400 rpm on an Eppendorf thermomixer for 10 min    -   magnetic separation and discarding of the supernatant    -   resuspend MagBead sediment in 100 μl of buffer AW2 (QIAGEN) and        transfer to a PCR tube    -   magnetic separation and discarding of the supernatant    -   rinsing of the bead under separated state with 3×150 μl of        double-distilled water    -   carefully and quantitatively remove supernatant    -   resuspend beads with 50 μl of PCR solution and start PCR1

Nested PCR1:

-   -   -   25 μl of Hot Star Tag Master Mix (QIAGEN)        -   1 μl of EOF (100 μM)        -   1 μl of EOR (100 μM)        -   23 μl of water        -   Tag activation 15 min; 95° C.

Cycle:

-   -   -   denaturation 15 s;        -   annealing 30 s; 52° C.        -   extension 15 s; 72° C.        -   number of cycles 20

    -   After PCR1: place PCR tube on the magnetic separations rack and        transfer 5 μl to a fresh PCR tube

    -   performing PCR2 (quantitative real-time PCR) by addition of 20        μl of PCR 2 reagents        -   12.5 μl of QIAGEN QuantiTect sample PCR Malta mix. 0.1 μl of            EFwd; 0.10 μl of ERev; 0.05 μl of EPro, 7.25 μl of            double-distilled water; cycling: Tag activation 5 min; 95°            C.; 40 cycles [denaturation 15 s; 95° C.; extension 60 s;            60° C.]

FIG. 4 clarifies the result of the quantitative PCR for the detection ofE. coli DNA from exemplary embodiment 4 with a different number ofbacteria in the preparation. In addition to the use of anion exchangemagnetic particles, it was also shown that the inventive process can inprinciple also be performed using silica magnetic particles andchaotropic nucleic acid binding.

In the course of a fifth exemplary embodiment, the RNA isolation and itsdetection are to be determined by means of the RNA bacteriophages “fr”.

For this, 500 μl of phage fr suspension [5×10⁶ PFU (plaque formingunits)] and 1 ml of lysis buffer GE (01% PVP MW 10,000; 0.45% Tween-20;0.45% NP-40; 10 mM tris/Cl pH 7.5; 1 mM EDTA), 20 μl of proteinase K and20 μl of AX01 beads were mixed in a 1.5 ml microtube.

First, the reagent vessel is incubated on an Eppendorf thermomixer at56° C. and 1400 rpm for 15 min. After a magnetic separation, thesupernatant is discarded and the magnetic particles are washed twicewith buffer GE (800 μl each; briefly vortex; magnetic separation anddiscarding of the supernatant). The magnetic particle sediment is nowresuspended in 1× QuantiTect sample RT-PCR Master Mix (QIAGEN GmbH) andtransferred to a PCR tube. After a further magnetic separation, thesupernatant is again quantitatively removed and the reaction is startedby addition and resuspension of the sediment in 50 μl of RT-PCRsolution.

The RT-PCR mix is composed as follows:

-   -   25 μl of 2× Quantitect sample RT-PCR Master Mix    -   0.5 μl of FOF (CTCGAAGTTTACCAATCAAT; 10 μM)    -   0.5 μl of FOR (TATTTATCTGACCACAACGG; 10 μM)    -   0.5 μl of RT mix    -   23.5 μl of water

For the RT-PCR the following temperature adjustments were utilized

-   -   Reverse transcription 35 min; 50° C.    -   Tag activation 15 min; 95° C.

Cycle:

-   -   denaturation 30 s; 95° C.    -   annealing 60 s; 52° C.    -   extension 60 s; 72° C.    -   number of cycles 40

After the RT PCR, 4 μl of supernatant of the 50 μl reaction were appliedto a 2% strength agarose gel and separated eleotrophoretically. Afterstaining of the gel with ethidium bromide, a recording was made on a geldocumentation device.

FIG. 6 shows on the agarose gel (2% strength) that both samples of thefr-phages isolation exhibit a specific band (middle and right trace)having the expected size (202 bp). Thus this amplification based on RNAas starting material can be rated as successful. A single RT-PCR cansuccessfully detect 5×10⁶ PFU of phage fr after the RNA preparation andamplification. The left trace shows the DNA lengths standard)

This and the sixth exemplary embodiment clearly show that the processaccording to the invention can be employed for the purification(isolation) and amplification of nucleic acids (as described in detailabove), in addition to the isolation and amplification of DNA, just aswell for the isolation and amplification of RNA. We refer to the abovedisclosure.

In the course of a sixth exemplary embodiment, HeLa cells were employedfor the isolation of RNA.

The following materials were employed:

-   -   Cultured HeLa cells (human cervical carcinoma cell line).    -   The cells were cultured in cell culture flasks by means of        standard cell culture protocols. After washing with PBS, the        cells were trypsinized and the cell count was determined under        the microscope.    -   AX1 anion exchange beads    -   lysis buffer GE (01% PVP MW 10,000; 0.45% Tween-20; 0.45% NP 40;        10 mM tris/Cl pH 7.5; 1 mM EDTA)

The procedure was according to the following protocol:

-   -   500 μl of 10⁵, or 10³ Hela cells in PBS are mixed with 1 ml of        buffer GE, 20 μl of proteinase K and 20 μl of AX1 MagBeads in a        2.2 ml microtube    -   Incubation: 15 min on an Eppendorf thermomixer at 56° C. and        1400 rpm    -   magnetic separation and discarding of the supernatant    -   wash Mag Beads 2× with buffer GE (800 μl each; vortex briefly;        magnetic separation and discarding of the residue)    -   resuspend MagBead sediment in 100 μl of double-distilled water        and transfer to a PCR tube    -   carefully and quantitatively remove supernatant    -   resuspend beads using 50 μl of RT-qPCR solution and start PCR in        a real-time cycler

For the RT-qPCR, the following temperature adjustments were utilized

-   -   25 μl of 2× QuantiTect sample RT-PCR Master Mix    -   0.5 μl of Fwd primer (human lamin A; GGCGGGTGGATGCTGAGAACA; 10        μM)    -   0.5 μl of Rev primer (human lamin A; TGTCAATCTCCACCAGTCGGG; 10        μM)    -   0.25 μl of sample (human lamin A; ATCTACAGTGAGGAGCTGCGTGAGA        (5′-Fam; 3′-BHQ1; 10 μM)    -   0.5 μl of RT mix    -   23.25 μl of water

For the RT-PCR, the following temperature adjustments were utilized

-   -   Reverse transcription 30 min; 50° C.    -   Taq activation 15 min; 95° C.

Cycle:

-   -   denaturation 15 s; 94° C.    -   annealing/extension 60 s; 60° C.    -   number of cycles: 40

FIG. 7 shows an “amplification plot” of a real-time PCR. The curve showsthe increase in the fluorescence of two RT QPCR amplifications of HeLaRNA preparations as a function of the number of the PCR cycle and thusclear amplification signals that prove that, using the processdescribed, RNA was isolated from HeLa cells, since the primer/samplesequences of the amplified lamin A gene are chosen such that because ofexon/intron structures of the gene, an amplification can start onlystarting from a spliced RNA. The example moreover also shows that undercertain circumstances a second amplification can also be dispensed with.

The process can easily be performed in a closed system in order thus toavoid contamination. In FIG. 5, an example of a closed system forperforming the process is shown in section. The device shown comprises alysis chamber 1, in which magnetic beads (in addition to proteinase K) 2for the binding of nucleic acid are already located. The lysis chamber 1has a sealable opening 3, by means of which a biological sample isintroduced into the lysis chamber 1. The lysis chamber 1 is providedwith a pressure equalization valve 4 in order to be able to regulate thepressure prevailing in the lysis chamber 1, or to empty this if requiredby means of overpressure. In the lysis chamber is situated a rod-shapedmagnet 5, with which the lysis can be mechanically assisted. If themagnet rotates rapidly enough, the magnetic beads separate with thebound nucleic acids and can be conducted further. Particularly simply,however, the lysis can also be assisted mechanically in this case withthe aid of ultrasound.

In the lysis chamber 1, the sample is digested by pumping a lysis bufferinto the lysis chamber via a connection 7, a channel 8, a valve unit 9,and a channel 10 from below via an opening 11. The sample digestedenzymatically by the lysis buffer and with stirring binds to themagnetic beads by anion exchange. After binding, the lysis buffertogether with the magnetic beads and the nucleic acids bound thereto areaspirated through the opening 11 and pumped through the channel 10 tothe valve unit 9 and from here led out via a channel 12 into a first PCRchamber 13. In the first PCR chamber 13 is situated a small permanentmagnet 14, to which the magnetic beads 2 adhere. The lysis buffer is ledfurther through a channel 15, a valve unit 16 and a channel 17 into awaste chamber 18. The waste chamber 18 has an outwardly leading opening19 in order thus to avoid the formation of an overpressure in the wastechamber 18. The connection 19 can be a valve or in the simplestembodiment a semipermeable membrane (e.g. GoreTex™),

Via the connection 7, wash buffers are now introduced through thechannel 8, the valve unit 9 and the channel 12 into the first PCRchamber and led further via the channel 15, the valve unit 16 and thechannel 17 into the waste chamber 18 in order thus to wash the magneticbeads with the nucleic acids bound thereto. Finally, the further supplyof wash buffers is stopped and the wash buffers are pumped out of thefirst PCR chamber. Water is now introduced into the system via aconnection 20. The water passes through a channel 21 into the valve unit16 and is introduced from this into a reagent chamber 23 via a channel22. In the reagent chamber are situated dried reagents 24 in order tocarry out a first PCR. The reagents 24 dissolve in the water and passthrough a channel 25, the valve unit 9 and the channel 12 into the firstPCR chamber 13. Here, a first, relatively unspecific amplification ofnucleic acid is performed. After the amplification in the first PCRchamber 13, an aliquot of the PCR reaction solution is introduced viathe channel 15 into a measuring channel 26 of the valve unit 16 furthervia channel 17 into the waste chamber 18. The valve unit 16 is now shutsuch that water can he introduced via a connection 20, and is led viathe channel 21 and the valve unit 16 and the measuring channel 26 withthe specified volume of PCR reaction solution containing the amplifiednucleic acid strands situated therein, directly further via a channel27, to a second PCR chamber 28.

In the second PCR chamber 28 are situated reagents 29 for performing asecond PCR, in which the amplification is very specific. The second PCRchamber 28 is connected to a channel 30, which externally comprises aconnection 31, which can be present in the form of a valve. By means ofthe connection 31, a pressure equalization is made possible in thesecond PCR chamber 28.

The device shown in FIG. 5 comprises a channel 32 that connects the twovalve units 9 and 16 to one another. This channel 32 is needed for theresuspension of the dried reagents in the chamber 23. For theresuspension, water flows via the access 20 and the channel 21 into thevalve unit 16 and is then led through the channel 32 into the valve unit9. From there, the water flows further through the stations 25, 23 and22 to the valve unit 16 and then further through the channel 15 to thefirst PCR chamber 13, which is thus filled from below. Deaeration of thechamber takes place via the stations 12 and 9 and the channel 30 via theelement 31.

The device shown in FIG. 5 can be designed as a disposable article,since this can be manufactured from inexpensive materials and in smallform. For performing the process, this device is employed in a furtherdevice that provides for an automated performance of the process. Thefurther device then actuates and controls the valve units (9, 16)automatically, and also the supply of buffers and water.

The specific amplification in the second PCR chamber can be detected bymeans of real-time fluorescence detection or by high-resolution DNA meltcurve analysis. For both types of detection, an optical device forfluorescence detection is intearated into the second PCR chamber 28.

1. A process for the preparation of a biological sample having thesteps: digestion of the biological sample; binding of the nucleic acidof the digested sample; non-specific amplification of the bound nucleicacids, in particular by means of a first PCR of a nested PCR; specificamplification of the nucleic acid strands resulting from the firstamplification, in particular using a second PCR of the nested PCR. 2.The process as claimed in claim 1, in which the bound nucleic acids arewashed before amplification.
 3. The process, as claimed in claim 1, inwhich nucleic acids obtained by digestion are bound to particles, theparticles with the nucleic acids bound thereto are separated and theseparated, bound nucleic acids are amplified.
 4. The process as claimedin claim 1, in which nucleic acids are bound to magnetic particles andbefore amplification of the nucleic acids the particles with the nucleicacids bound thereto are bound magnetically using a magnet and the magnetwith the magnetic particles situated thereon is separated beforeamplification.
 5. The process as claimed in claim 1, in which followingthe first non-specific amplification the particles are separated andonly the supernatant thus obtained is supplied completely or partiallyto the second PCR of a nested PCR.
 6. The process as claimed in claim 1,in which a lysis buffer employed for the digestion has a salt content ofin total less than 50 millimolar.
 7. The process as claimed in claim 6,in which the lysis buffer has a pH of 7.5 to 10.5, the pH preferablybeing stabilized by the buffers tris, hepes and/or mops.
 8. The processas claimed in claim 6, in which the lysis buffer comprises nonionicdetergents, namely preferably 0.05 to 0.4% by volume.
 9. The process asclaimed in claim 6, in which the lysis buffer comprises PVP, in factpreferably up to 0.1% by volume.
 10. The process as claimed in claim 1,the process being performed in a closed system which comprises a lysischamber and two chambers, the first non-specific amplification beingperformed in the first chamber and the second specific amplificationbeing performed in the second chamber.
 11. A device for performing aprocess as claimed in claim 1, having a lysis chamber, a first PCRchamber and connecting means for connecting the lysis chamber to thefirst PCR chamber, with a magnet in the first PCR chamber, and a secondPCR chamber, and connecting means for connecting the first PCR chamberto the second PCR chamber.
 12. The device as claimed in claim 11, havingreagents for performing a second specific amplification, which arelocated in the second PCR chamber.
 13. The device as claimed in claim11, having reagents that are located in a reagent chamber, having meansfor connecting the reagent chamber to the first PCR chamber and meansfor supplying water to the reagent chamber.
 14. The device as claimed inclaim 11, containing magnetic particles in the lysis chamber for bindingnucleic acids.
 15. The device as claimed in claim 11, having a wastechamber and means for connecting the first PCR chamber to the wastechamber.
 16. The device as claimed in claim 11, having an anionexchanger for binding the nucleic acid.
 17. The device as claimed inclaim 11, having an ultrasound source for carrying out the lysis.